The plasma membrane (PM) of polarized epithelial cells is physically continuous, but functionally and compositionally divided into two separate domains: the apical and basolateral. The molecular sorting mechanisms and pathways by which newly synthesized PM proteins achieve their specific yet asymmetric distributions are actively being examined in many polarized epithelial cell types. Newly synthesized apical proteins are delivered to the apical PM by two pathways: the direct or indirect. Proteins using the direct pathway are delivered directly form the trans-Golgi network (TGN) to the apical surface whereas proteins using the indirect pathway take a more circuitous route. They are first delivered from the TGN to the basolateral domain where they are retrieved by endocytosis and transcytosed to the apical surface. This proposal focuses on the role of MAL l and MAL2 proteolipids in regulating apical delivery. MAL l has been implicated in sorting proteins in the direct pathway and MAL2 has a presumed role in indirect sorting. Our long-term goal is to understand the mechanisms regulating apical protein delivery in hepatocytes, the major epithelial cell of the liver. Unlike most simple epithelial cells, hepatocytes use the indirect pathway for apical protein delivery. We have previously shown that indirect sorting in hepatocytes requires cholesterol and glycosphingolipids. Because MAL l and MAL 2 have been identified as important regulators of apical PM delivery in both pathways and because their activity requires cholesterol and glycosphingolipids, we propose to characterize the mechanism by which the MAL proteolipids function in apical PM sorting in polarized hepatic cells in Aim 1 and in non-polarized hepatic cells in Aim 3. Interestingly, the tissue expression patterns of MAL l and MAL 2 are generally non-overlapping suggesting they participate in cell-specific transport pathways. Does MAL2 confer and regulate indirect sorting of apical proteins in hepatocytes? Likewise, does MAL l confer direct apical targeting from the TGN in kidney-derived cells? We intend to test this hypothesis in Aim 2. Identifying the mechanisms regulating the vesicle-mediated delivery of newly synthesized apical residents to the apical PM is imperative to understanding how this surface is established and maintained. Such information will provide insight into the molecular basis of human liver disease and other diseases of epithelial cells.